The long-term aim of this project is to understand the function(s) of modified nucleotides (predominantly N6-methyladenosine) located internally in most mRNAs of higher eukaryotes. Retrovirus RNAs will be studied in detail with most emphasis placed on analysis of the genomic and intracellular RNAs of Rous sarcoma virus. Several techniques will be explored for localization of m6A on RNAs, including use of specific antibodies against m6A, which will be visualized by electron microscopy, as well as used for selection of RNAs containing m6A; blotting techniques in which RNAs labeled specifically at methylated nucleotides will be hybridized to restriction fragments of viral DNA obtained from molecular clones; and both rapid and more classical RNA sequencing techniques. I intend to localize m6A to the nucleotide level in the RSV genomic RNA and to compare the approximately 12 methylated sites here with those in RSV nuclear and specific mRNA species. The relationship of methylated bases to sites of RNA processing, translational initiation and termination sites, reverse transcription, RNA packaging, or to RNA stability or secondary structure will be assessed. The degree of conservation of methylated sites will be studied by use of deletion mutants and by comparisons between different RSV strains, between ALV and MuLV, and between RSV and PRC II ASV. In addition, modification of the cellular src mRNA will be compared with that in the viral src mRNA and in the RNA genome. Further, the effects on RNA modification of growing RSV in avian or mammalian hosts will be compared. In addition, I will perturb the normal methylation pattern of RSV RNA by use of inhibitors of methylation, hypermethylation of the RNA, and site-directed mutagenesis to alter the normal sites of methylation. The effects of these alterations on retrovirus replication and transformation will be studied.